WO2002092132A2 - Microparticules et methodes d'apport de vaccins a l'aide de virus de recombinaison - Google Patents

Microparticules et methodes d'apport de vaccins a l'aide de virus de recombinaison Download PDF

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Publication number
WO2002092132A2
WO2002092132A2 PCT/US2002/000235 US0200235W WO02092132A2 WO 2002092132 A2 WO2002092132 A2 WO 2002092132A2 US 0200235 W US0200235 W US 0200235W WO 02092132 A2 WO02092132 A2 WO 02092132A2
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viral vector
microparticle
virus
antigen
cell
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PCT/US2002/000235
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WO2002092132A3 (fr
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John Hural
Mark E. Johnson
A. Gregory Spies
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Corixa Corporation
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Publication of WO2002092132A2 publication Critical patent/WO2002092132A2/fr
Publication of WO2002092132A3 publication Critical patent/WO2002092132A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10351—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/70—Vectors containing special elements for cloning, e.g. topoisomerase, adaptor sites
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to formulations, compositions and methods that can be used for the delivery of vaccines comprising virus particles, virus-like particles or virus replicon particles conjugated to microparticles. More particularly, the virus particles, virus-like particles or virus replicon particles include a polynucleotide that encodes an immunogenic polypeptide.
  • Recombinant virus-mediated gene transfer is an attractive method of gene delivery to some cell types, particularly in vitro.
  • Advantages of viral systems include the large carrying capacity for recombinant transgenes, viral components that direct the genetic material to the nucleus and initiate transcription, and the ability to grow and purify high titers of the vector.
  • the utilization of viruses alone as an in vivo delivery system for the induction of immune responses has limitations.
  • One such limitation is the cellular tropism of the virus.
  • APCs efficiendy infect antigen presenting cells
  • DCs dendritic cells
  • the invention provides a viral vector conjugated to a microparticle, wherein the viral vector comprises a polynucleotide encoding a heterologous lrnmunogenic polypeptide. Conjugation of the viral vector to the microparticle results in a dramatic increase in the efficacy of the elicited immune response.
  • the viral vector can comprise a virus particle, a virus-like particle or a virus replicon particle.
  • the microparticle can be con j ugated to the viral vector by covalent interaction or by non-covalent interaction.
  • the viral vector is derived from a virus that enters cells via receptor mediated endocytosis, such as a rhinovirus, adenovirus, adeno-associated virus, enterovirus, pokovirus, coxsackie virus, echovirus, cardiovirus, hepatovirus, alphavirus, rubellavirus, flavivirus, pestivirus, hepatitis C virus, orthomyxovirus, bunyavirus, hantavirus, or nairovirus.
  • the viral vector is derived from a virus that enters cells via pH independent membrane fusion, such as a parainfluenza virus, mumps virus, measles virus or respiratory syncytial virus.
  • the microparticle has a characteristic length of about 0.5 ⁇ m to about 20 ⁇ m.
  • the microparticle comprises a positively charged surface.
  • the positive charge may be due to characteristics of the wall-forming material itself, or to an additive that is added to the polymer-solvent solution and/or to the process media used in the preparation of the microparticles.
  • the microparticle can further comprise a cationic lipid, a polymer of a natural or synthetic monomer, or an anionic surfactant.
  • the microparticle comprises polyvinyl alcohol, polyvinyl pyrilidone, carboxymethyl cellulose, gelatin, or polyoxyethylene(20) sorbitan monolaurate (e.g., TweenTM 20, TweenTM 80; Sigma-Aldrich Corp., St. Louis, MO).
  • polyvinyl alcohol polyvinyl pyrilidone
  • carboxymethyl cellulose gelatin
  • polyoxyethylene(20) sorbitan monolaurate e.g., TweenTM 20, TweenTM 80; Sigma-Aldrich Corp., St. Louis, MO.
  • the invention additionally provides a method for delivering a polynucleotide to a cell comprising contacting the cell with a microparticle of the invention.
  • the cell is an antigen-presenting cell, such as a dendritic cell.
  • the contacting can occur ex vivo or in vivo.
  • the heterologous polypeptide is an antigen or other immunogenic molecule.
  • the antigen can be associated with cancer, autoimmune disease or infectious disease. In one embodiment, the antigen is associated with M. tuberculosis.
  • the invention further provides a vaccine comprising a microparticle of the invention and, optionally, further comprising an adjuvant.
  • the invention thus provides a method for delivering a polynucleotide to a subject comprising a(_lministering to the subject a vaccine of the invention. Also provided is a method of stimulating an immune response in a subject, a method of treating cancer in a subject, a method of inhibiting tumor growth in a subject, a method of treating autoimmune disease in a subject, and a method of treating an infection in a subject.
  • Figure 1 is a graph showing proliferation, as measured by incorporation of 3 H- thymidine (in counts per minute, CPM), of 1-lB T cells in response to stimulation with dendritic cells that had been exposed to recombinant adenovirus encoding the M. tuberculosis antigen 38-1 (Adeno/38-1), Adeno/38-1 conjugated to microparticles of the invention (Adeno/38-1 +Microparticles), Adeno/38-1 mixed with hpofectamine (Adeno/38-1 +Lipofectamine), or Peptide 2-11, which is the minimal peptide recognized by 1-lB T cells.
  • Adeno/38-1 Adeno/38-1 conjugated to microparticles of the invention
  • Ado/38-1 +Microparticles Adeno/38-1 mixed with hpofectamine
  • Peptide 2-11 which is the minimal peptide recognized by 1-lB T cells.
  • Figure 2 is a graph showing interferon gamma (IFN- ⁇ ) production, measured as optical density (O.D.) at 450-570 nm, by 1-lB T cells in response to stimulation with dendritic cells that had been exposed to recombinant adenovirus encoding the M. tuberculosis antigen 38-1 (Adeno/38-1), Adeno/38-1 conjugated to microparticles of the invention (Adeno/38-1 + Microparticles), Adeno/38-1 mixed with lipofectamine (Adeno/38-1 +Lipofectamine), or Peptide 2-11, which is the minimal peptide recognized by 1 -lB T cells.
  • IFN- ⁇ interferon gamma
  • Figure 3 is a graph showing interferon gamma (IFN- ⁇ ) production, measured as optical density (O.D.) at 450-570 nm, by 1 -lB T cells in response to stimulation with dendritic cells that had been exposed to recombinant adenovirus encoding the M.
  • tuberculosis antigen TbH9 TbH9 ad
  • microparticles made with polymer formulation A TC339)
  • TbH9 ad conjugated to formulation A microparticles TbH9+TC339)
  • recombinant adenovirus encoding the M recombinant adenovirus encoding the M.
  • tuberculosis antigen 38-1 38-1 (38-1 ad), 38-1 ad conjugated to formulation A microparticles (38-1 ad+TC339), 38-1 ad conjugated to a second lot of formulation A microparticles (38-1 ad+TC350b), 38-1 ad conjugated to formulation C microparticles (38-1 ad+CD125), or 38-1 ad conjugated to formulation B microparticles (38-1 ad+CD165b).
  • Figure 4 is a graph showing interferon gamma (IFN- ⁇ ) production, measured as optical density (O.D.) at 450-570 nm, by 1 -lB T cells in response to stimulation with dendritic cells that had been exposed to recombinant M. tuberculosis antigen 38-1 (r38-l), r38-l conjugated to microparticles made with formulation A (r38-l +TC339), r38-l ad conjugated to microparticles made with a second lot of formulation A (r38-l +TC350b), or r38-l conjugated to formulation C microparticles (r38-l +CD125).
  • IFN- ⁇ interferon gamma
  • Figure 5 is a graph showing interferon gamma (IFN- ⁇ ) production, measured as optical density (O.D.) at 450-570 nm and plotted as a function of multiplicity of infection (MOI), by DPV specific murine CD8+ T cells in response to stimulation with DC2.4 antigen presenting cells that had been infected 24 hours prior with either a recombinant DPV adenovirus or a control recombinant adenovirus (TbH9 adeno).
  • the adenovirus was added naked, or following preincubation with lipfectamine (lipo) or was pre-adsorbed to PLG microspheres (ms). After 48 hours of culture, supernatants were collected for the assessment of IFN- ⁇ production.
  • FIG. 6 is a graph showing T cell proliferation (CPM incorporated) as a function of multiplicity of infection (MOI) for cells prepared as described for Figure 5, with the exception of the final step. After 48 hours in culture, the plates were pulsed with tritiated thymidine to measure the proliferation of T cells.
  • Figures 7A-C are photomicrographs showing that conjugation of adenovirus to cationic microparticles augments infection of human DC: Adenovirus expressing hrGFP was added to 2.5 x 10 5 human DC alone (naked; 7A), treated with lipofectamine (7B) or conjugated to microparticle formulation A (7C) at an MOI of 20 and incubated for 16hrs at 37°C. hrGFP expression was visualized by fluorescence microscopy at lOx magnification.
  • Figures 8A-D are photomicrographs showing that conjugation of adenovirus to cationic microparticles allows detectable expression at low MOI: Adenovirus expressing hrGFP was conjugated to formulation A and subjected to 1/5 serial dilution. The diluted conjugates were added to 2.5 x 10 5 human DC per well at an MOI of 100 (8A), 20 (8B), 4 (8C) or 0.8 (8D) and incubated 16hrs at 37°C. Expression was visualized by fluorescence microscopy at 1 Ox magnification.
  • the invention is based on the discovery that delivery of combinations of viral vectors and microparticles facilitate the infection of phagocytic antigen-presenting cells (APCs).
  • APCs phagocytic antigen-presenting cells
  • Data described herein show that adenovirus, which alone does not efficiendy infect dendritic cells, does infect dendritic cells more effectively when combined with microparticles.
  • These infected cells subsequentiy present the virally encoded heterologous antigen to T cells in a productive manner.
  • a far more effective immune response can be elicited when viral vectors encoding immunogenic polypeptides are conjugated to microparticles.
  • This invention therefore provides compositions and methods for delivery of immunogenic molecules that offers the advantages of viral delivery systems while also overcoming problems encountered with delivery using virus alone.
  • nucleic acid or “polynucleotide” refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.
  • polypeptide includes proteins, fragments of proteins, and peptides, whether isolated from natural sources, produced by recombinant techniques or chemically synthesized. Polypeptides of the invention typically comprise at least about 8 amino acids.
  • conjugation refers to the joining together of elements, such as by covalent or non-covalent interactions.
  • covalent interactions include chemical bonds; and examples of non-covalent interactions include ionic interactions, van der Waals forces, and hydrophobic and hydrophilic interactions.
  • conjugation of a viral vector to a microparticle is surface adsorption of the viral vector to the microparticle.
  • viral vector means a virus particle, virus-like particle, virus replicon particle, or an equivalent thereof, that includes a polynucleotide encoding a polypeptide that is not native to the virus.
  • an "immune response” is evidenced by conventional indicators of a protective immune response, including, but not limited to, release of gamma interferon (IFN- ⁇ ), T cell proliferation, and cytokine or antibody production.
  • IFN- ⁇ gamma interferon
  • microparticle refers to a material comprising a wall forming material and having surface charge characteristics, size and morphology capable of delivering a viral vector into a cell. Microparticles may be solid or porous, have a rough or smooth surface, and may have a regular or irregular shape. Examples of microparticles include, but are not limited to, microspheres, sheets, rods and tubes.
  • characteristic length means length, width or diameter of a microparticle, as appropriate given the morphology of the relevant microparticle.
  • subject refers to the recipient of the therapy to be practiced according to the invention.
  • the subject can be any vertebrate, but will preferably be a mammal. If a mammal, the subject will preferably be a human, but may also be a domestic livestock, laboratory subject or pet animal.
  • to "prevent” a disease or condition means to hinder or delay the onset or development of the disease or condition.
  • to "treat" a disease or condition means to ameliorate one or more symptoms associated with the disease or condition.
  • antigen-presenting cell means a cell capable of handling and presenting antigen to a lymphocyte.
  • APCs include, but are not limited to, macrophages, Langerhans-dendritic cells, follicular dendritic cells, B cells, monocytes, fibroblasts and fibrocytes.
  • Dendritic cells are a preferred type of antigen presenting cell. Dendritic cells are found in many non-lymphoid tissues but can migrate via the afferent lymph or the blood stream to the T-dependent areas of lymphoid organs. In non- lymphoid organs, dendritic cells include Langerhans cells and interstitial dendritic cells. In the lymph and blood, they include afferent lymph veiled cells and blood dendritic cells, respectively. In lymphoid organs, they include lymphoid dendritic cells and interdigitating cells.
  • modified to present an epitope refers to antigen-presenting cells (APCs) that have been manipulated to present an epitope by natural or recombinant methods.
  • APCs antigen-presenting cells
  • the APCs can be modified by exposure to the isolated antigen, alone or as part of a mixture, peptide loading, or by genetically modifying the APC to express a polypeptide that includes one or more epitopes.
  • salts refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects.
  • examples of such salts include, but are not limited to, (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, furmaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; (b) salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobal
  • pharmaceutically acceptable carrier includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system.
  • examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/ water emulsion, and various types of wetting agents.
  • Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline.
  • Compositions comprising such carriers are formulated by well known conventional methods (see, for example, Remington's Pharmaceutical Sciences, Chapter 43, 14th Ed., Mack Publishing Co, Easton PA 18042, USA).
  • adjuvant includes those adjuvants commonly used in the art to facilitate the stimulation of an immune response.
  • adjuvants include, but are not limited to, helper peptide; aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; Freund's Incomplete Adjuvant and Complete Adjuvant (Difco
  • AS-2 Smith-Kline Beecham
  • QS-21 Aminomycin
  • MPLTM immunostimulant or 3d-MPL Comixa Corporation
  • LEIF salts of calcium, iron or zinc
  • an insoluble suspension of acylated tyrosine acylated sugars
  • acylated sugars cationically or anionically derivatized polysaccharides
  • polyphosphazenes aminoalkyl glucosaminide phosphate (AGP)
  • isotucaresol monophosphoryl lipid A and quil A
  • muramyl tripeptide phosphatidyl ethanolamine or an irrirnunostimulating complex including cytokines (e.g., GM-CSF or interleukin-2, -7 or —12) and i_____munos_imulatory DNA sequences.
  • cytokines e.g., GM-CSF or interleukin-2, -7 or —12
  • an adjuvant such as a helper peptide or cytokine can be provided via a polynucleotide encoding the adjuvant.
  • the invention provides a polynucleotide delivery system comprising one or more vectors conjugated to a microparticle.
  • the vector is a viral vector, such as a virus particle, a virus-like particle, a virus replicon particle or an equivalent of any one of the foregoing.
  • the vector comprises a polynucleotide encoding a heterologous, immunogenic polypeptide that is capable of eliciting or enhancing an immune response.
  • Microparticle morphology can include spheres, sheets, rods, tubes and other shapes, and be solid or porous.
  • the microparticles can have smooth surfaces, angular surfaces, rough surfaces, porous surfaces, or sharp edges.
  • Microparticle size can vary over a fairly broad range, e.g., from about 0.2 ⁇ m to about 40 ⁇ m in diameter or length, and still be effective.
  • the microparticles are about 0.5 ⁇ m to about 20 ⁇ m in diameter or length.
  • the microparticle diameter or length is about 1 to about 10 ⁇ m.
  • Microparticles in this size range are well-suited to be phagocytosed by antigen- presenting cells, leading to effective T cell stimulation.
  • the microparticle material can comprise any of a wide range of particles, including such exemplary wall forming materials as described in U.S. Patent No. 5,407,609. Biocompatible materials are preferred for uses that involve administration to patients. Biodegradable materials are also preferred.
  • biodegradable polymers such as poly(lacto-co-glycolide) (PLG), poly(lactide), poly(glycolide), poly(caprolactone), poly(hydroxybutyrate) and/or copolymers thereof.
  • the microparticles can comprise another wall-forming material.
  • Suitable waU-forming materials include, but are not limited to, poly(dienes) such as poly(butadiene) and the like; poly(alkenes) such as polyethylene, polypropylene, and the like; poly(acrylics) such as poly(acrylic acid) and the like; poly(methacrylics) such as poly(methyl methacrylate), poly(hydroxyethyl methacrylate), and the like; poly(vinyl ethers); poly (vinyl alcohols); poly (vinyl ketones); poly (vinyl halides) such as poly (vinyl chloride) and the like; poly (vinyl nittiles), poly (vinyl esters) such as poly(vinyl acetate) and the like; poly(vinyl pyridines) such as poly(2-vinyl pyridine), poly(5-methyl-2-vinyl pyridine) and the like; poly(carbonates); poly(esters); poly(orthoesters); poly(esteramides); poly
  • Biodegradable microsphcres for use as carriers are disclosed, for example, in U.S. Patent Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344; 5,407,609; and 5,942,252; the disclosures of each of which are incorporated herein by reference.
  • these patents such as U.S. Patent No. 4,897,268 and 5,407,609, describe the production of biodegradable microspheres for a variety of uses, but do not teach the optimization of microsphere formulation and characteristics for DNA delivery.
  • the invention provides a method for producing a microparticle, as well as a method for producing a viral vector con j ugated to a microparticle.
  • the method for producing a microparticle comprises dissolving a polymer in a solvent to form a polymer solution.
  • a cationic compound such as DOTAP
  • DOTAP can be added to the solvent.
  • the polymer solution is then emulsified and diluted.
  • the microparticles are hardened
  • the method can further comprise subsequent steps of washing, freezing and lyophilizing the microparticles.
  • a double-emulsion techmque can be used.
  • the microparticle comprises a cationic hpid, a polymer of a natural or synthetic monomer, or an anionic surfactant.
  • the microparticle comprises a positively charged surface. Both cationic and anionic components can be introduced into the microparticles to manipulate surface charge.
  • a positive surface charge for example, can be created by selection of a wall-forming material that will impart positive charges on the surface, such as polylysine, modified PLG, for example.
  • additives such as DOTAP or other charged compounds, can be added to the polymer-solvent solution and/or to the process media, to alter the surface charge of the resulting microparticles.
  • the polymer comprises PLG.
  • the PLG can include ester end groups or carboxylic acid end groups, and have a molecular weight of from about 4 kDa to about 120 kDa, or preferably, about 8 kDa to about 65 kDa.
  • the solvent can comprise, for example, dichloromethane, chloroform, or ethylacetate.
  • the polymer solution further comprises a cationic hpid and/or an adjuvant, such as MPL.
  • stabilizers include, but are not limited to, carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), or a mixture thereof.
  • the stabilizer can optionally further comprise a cationic hpid.
  • the stabilizer comprises from about 0 to about 10% of the process medium, or preferably, about 1% to about 5% of the process medium.
  • the solvent comprises an internal water volume of from about 0.001% to about 0.5%; and/or the aqueous solution comprises an ethanol content of from about 0% to about 75% (v/v).
  • the solvent used to dissolve the wall material or excipient can be selected from a variety of common organic solvents including halogenated aliphatic hydrocarbons such as methylene chloride, chloroform, and the like; alcohols, aromatic hydrocarbons such as toluene and the like; halogenated aromatic hydrocarbons; ethers such as methyl t-butyl ether and the like; cyclic ethers such as tetrahydrofuran and the like; ethyl acetate; diethyl carbonate; acetone; cyclohexane; and water These solvents may be used alone or in combination.
  • halogenated aliphatic hydrocarbons such as methylene chloride, chloroform, and the like
  • alcohols aromatic hydrocarbons such as toluene and the like
  • halogenated aromatic hydrocarbons ethers such as methyl t-butyl ether and the like
  • cyclic ethers such as tetrahydrofuran and the like
  • the solvent chosen must be a material that will dissolve the wall material or excipient and it is best that it is chemically inert with respect to the polymer or other components of the viral dehvery system. Moreover, the solvent should have limited solubility in the extraction medium. Generally, limited solubility means having a solubility from about 1 part per 100 to about 25 parts per 100. Preferred solvents include ethyl acetate, diethyl carbonate, chloroform and methylene chloride.
  • the viral vector can comprise a virus particle, a virus-like particle or a virus replicon particle.
  • virus particles, virus-like particles and virus rephcon particles that normally enter a cell via receptor mediated endocytosis are particularly well-suited to this dehvery system due to their increased rate and efficiency of APC infection as well as their enhanced antigen presentation over virus alone. This includes a large variety of both enveloped and non- enveloped viruses. It is also likely that infection of phagocytic cells by some viruses that normally enter cells via other mechanisms, such as pH independent membrane fusion, will also be aided by conjugation with microparticles.
  • the virus is selected from those viruses that normally enter a cell by receptor mediated endocytosis.
  • the virus is selected from those viruses which have already been developed for expression of heterologous recombinant genes.
  • viruses that enter cells via receptor mediated endocytosis include, but are not limited to, rhinovirus, adenovirus, adeno-associated virus, enterovirus, poliovirus, coxsackie virus, echovirus, cardiovirus, hepatovirus, alphavirus, rubellavirus, flavivirus, pestivirus, hepatitis C virus, orthomyxovirus, bunyavirus, hantavirus, or nairovirus.
  • viruses that enter cells via pH independent membrane fusion include, but are not limited to, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, retrovirus, herpes virus and pox virus.
  • Microparticles and viruses can be combined for in vivo or in vitro use in a variety of manners including, but not limited to, pre-, post- lyophilization, solution/dry, buffer type and pH, excipients present, time and temperature of incubation, mixing conditions, ratios of particles and virus, and concentrations of particles and virus.
  • the microparticle can be conjugated to the viral vector by covalent interaction or by non-covalent interaction. Examples of covalent interactions include chemical bonds; and examples of non- covalent interactions include ionic interactions, van der Waals forces, and hydrophobic and hydrophihc interactions.
  • One example of conjugation of a viral vector to a microparticle is surface adsorption of the viral vector to the microparticle.
  • compositions that are useful for delivering polynucleotides.
  • the composition is a pharmaceutical composition.
  • the composition can comprise a therapeutically or prophylactically effective amount of a polynucleotide that encodes an immunogenic polypeptide.
  • An effective amount is an amount sufficient to elicit or augment an immune response, e.g., by activating T cells.
  • One measure of the activation of T cells is a cytotoxicity assay or an interferon-gamma release assay, as described in the examples below.
  • the composition is a vaccine.
  • the condition to be treated or prevented is cancer or a precancerous condition (e.g., hyperplasia, metaplasia, dysplasia).
  • cancer include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothehosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
  • the condition to be treated or prevented is an infectious disease.
  • infectious disease include, but are not limited to, infection with a pathogen, virus, bacterium, fungus or parasite.
  • viruses include, but are not limited to, hepatitis type B or type C, influenza, varicella, adenovirus, herpes simplex virus type I or type II, rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovicus, arbovirus, hantavirus, coxsachie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I or type II.
  • bacteria examples include, but are not limited to, M. tuberculosis, mycobacterium, mycoplasma, neisseria and legionella.
  • parasites examples include, but are not limited to, rickettsia and chlamydia.
  • compositions of the present invention can optionally include a carrier, such as a pharmaceutically acceptable carrier.
  • a carrier such as a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being a(__ministered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.
  • Formulations suitable for parenteral administration such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, and carriers include aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, preservatives, liposomes, microspheres and emulsions.
  • aqueous isotonic sterile injection solutions which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient
  • aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, preservatives, liposomes, micro
  • composition of the invention can further comprise one or more adjuvants.
  • adjuvants include, but are not limited to, helper peptide, alum, Freund's, muramyl tripeptide phosphatidyl ethanolamine or an irnmunostimulating complex, including cytokines.
  • an adjuvant such as a helper peptide or cytokine can be provided via a polynucleotide encoding the adjuvant.
  • a preferred adjuvant is AGP.
  • adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as hpid A, Bortadell pertussis or Mycob cte ⁇ u tuberculosis derived proteins.
  • Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes biodegradable microspheres; monophosphoryl hpid A and quil A. Cytokines, such as GM CSF or interleukin-2, -7, or -12, may also be used as adjuvants.
  • the adjuvant composition is preferably designed to induce an immune response predominantly of the Thl type.
  • High levels of Thl-type cytokines e.g., IFN- ⁇ , IL-2 and IL-12
  • Th2- type cytokines e.g., IL-4, IL-5, IL-6, IL-10 and TNF- ⁇
  • a patient will support an immune response that includes Thl- and Th2-type responses.
  • Thl-type cytokines will increase to a greater extent than the level of Th2-type cytokines.
  • the levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, 1989, Ann. Rev. Immunol. 7:145-173.
  • Preferred adjuvants for use in eliciting a predominandy Thl-type response include, for example, a combination of monophosphoryl hpid A, preferably 3-de-O-acylated monophosphoryl hpid A (3D-MPL), together with an aluminum salt.
  • MPL adjuvants are available from Corixa Corporation (Hamilton, MT) (see US Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094).
  • CpG-containing oligonucleotides in which the CpG dinucleotide is unmethylated also induce a predominandy Thl response.
  • oligonucleotides are well known and are described, for example, in WO 96/02555.
  • Another preferred adjuvant is a saponin, preferably QS21, which may be used alone or in combination with other adjuvants.
  • QS21 a monophosphoryl hpid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739.
  • compositions comprise an oil-in-water emulsion and tocopherol.
  • a particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.
  • Another adjuvant that may be used is AS-2 (Smith-Kline Beecham). Any vaccine provided herein may be prepared using well known methods that result in a combination of antigen, immune response enhancer and a suitable carrier or excipient.
  • Vaccine preparation is generally described in, for example, M.F. Powell and M.J. Newman, eds., "Vaccine Design (the subunit and adjuvant approach),” Plenum Press (NY, 1995).
  • Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive.
  • compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.
  • buffers e.g., neutral buffered saline or phosphate buffered saline
  • carbohydrates e.g., glucose, mannose, sucrose or dextrans
  • mannitol proteins
  • proteins polypeptides or amino acids
  • proteins e.glycine
  • antioxidants e.g., antioxidants, chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule or sponge that effects a slow release of compound following a ⁇ _lministration).
  • sustained release formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site.
  • Sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane.
  • Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
  • the invention provides a method for delivering a polynucleotide to a cell.
  • the method comprises contacting the cell with a polynucleotide dehvery system of the invention, such as a viral vector conjugated to a microparticle.
  • the contacting can occur ex vivo or in vivo.
  • the cell can be a patient's own cells, to be re-introduced to the patient after ex vivo contact with the dehvery system or to be contacted in vivo following adrninistration of the dehvery system to the patient.
  • the contacting can occur in an ex vivo environment for any purpose in which polynucleotide dehvery into a cell is desired.
  • the cell is an antigen-presenting cell, such as a dendritic cell.
  • other cells, capable of expressing the polynucleotide are used.
  • the invention provides a method for delivering a polynucleotide to a subject.
  • the method comprises aclministering to the subject a polynucleotide dehvery system, or a composition, of the invention.
  • the invention further provides a method of stirnulating an immune response to an immunogenic polypeptide in a subject, a method of inhibiting tumor growth in a subject, a method of prolonging survival in a subject having a cancer, and a method of treating or preventing cancer, autoimmune disease, or infectious disease.
  • the method comprises administering to the subject a composition or dehvery system of the invention.
  • Treatment includes prophylaxis and therapy.
  • Prophylaxis or treatment can be accomphshed by a single direct injection at a single time point or multiple time points. Administration can also be nearly simultaneous to multiple sites.
  • Patients or subjects include mammals, such as human, bovine, equine, canine, feline, porcine, and ovine animals. Preferably, the patients or subjects are human.
  • compositions are typically administered in vivo via parenteral (e.g. intravenous, subcutaneous, and intramuscular) or other traditional direct routes, such as buccal/sublingual, rectal, oral, nasal, topical, (such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial, intraperitoneal, intraocular, or intranasal routes or direcdy into a specific tissue.
  • parenteral e.g. intravenous, subcutaneous, and intramuscular
  • other traditional direct routes such as buccal/sublingual, rectal, oral, nasal, topical, (such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial, intraperitoneal, intraocular, or intranasal routes or direcdy into a specific tissue.
  • the dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time, or to inhibit infection or disease due to infection.
  • the composition is administered to a patient in an amount sufficient to ehcit an effective immune response to the specific antigens and/or to alleviate, reduce, cure or at least partially arrest symptoms and/or complications from the disease or infection.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.”
  • the dose will be determined by the activity of the composition produced and the condition of the patient, as well as the body weight or surface areas of the patient to be treated.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side effects that accompany the administration of a particular composition in a particular patient.
  • the physician In determining the effective amount of the composition to be administered in the treatment or prophylaxis of diseases, the physician needs to evaluate the production of an immune response against the pathogen, progression of the disease, and any treatment-related toxicity.
  • Administration by many of the routes of administration described herein or otherwise known in the art may be accomphshed simply by direct adudinistration using a needle, catheter or related device, at a single time point or at multiple time points.
  • Example 1 Microparticle preparation
  • Microparticles were prepared using a variation on the double emulsion technique.
  • Poly(lactide-co-glycolide) (PLG) polymer (MW -40,000 Da) having ester end groups was dissolved in a solvent, dichloromethane, to a concentration of 33 mg/ml.
  • 25 mg of a cationic compound, ckoleoyl-l,2-diacyl-3-teime ylan ⁇ monium-propane (DOTAP) was also added to the solvent.
  • This mixture was then emulsified in 15 ml of an aqueous solution containing 5% polyvinyl alcohol (PVA) using a Silverson mixer. The dispersion was then diluted with 50 ml of 1% PVA and mixed for several hours.
  • PVA polyvinyl alcohol
  • the hardened microspheres were washed several times with distilled water, collected by centrifugation, and lyophihzed in the presence of mannitol. Particles were prepared in the absence of viral particles, and the end product was suitable for subsequendy being combined with the viral material.
  • This microparticle formulation is referred to herein as "Formulation A", and mcludes two lots, identified as TC339 and TC350b.
  • Formulation B is prepared in manner similar to that described above for Formulation A, except that the polymer used in formulation B has a mean molecular weight of about 8- 10 kDa and has acid end groups (rather than ester end groups). Two lots of formulation B are referred to herein as CD056 and CD150b.
  • a third microparticle formulation referred to as "Formulation C" has also been prepared.
  • Formulation C was prepared with PLG having an average molecular weight of about 40,000 Da and ester end groups, and dissolved in dichloromethane (DCM; 86 mg/ml).
  • DCM dichloromethane
  • 5.1 ml of aqueous solution was emulsified into this solution using a Polytron mixer for 20 seconds.
  • This primary emulsion was added to 280 ml of an aqueous solution of CMC (1.4%) and emulsified at 4500 for 75 seconds using a Silverson mixer.
  • the microparticles were mixed for several hours.
  • Example 2 Microparticle characterization
  • a Honba LA920 particle size analyzer was used to determine the size and size distribution of the microparticles. In addition, an aliquot was set aside for examination using scanning electron micrographs (SEM). The surface charge (zeta-potentia ⁇ ) of the microparticles was measured using a Malvern Zetasizer. The microparticles were also assayed for their ability to bind plasmid DNA to their surface. Briefly, a solution of plasmid DNA (3-10 kbp) was used to disperse the microparticles. After incubation, the microparticles were centrifuged, and the supernatant removed for analysis. DNA concentrations were quantified using the Picogreen DNA assay (Molecular Probes, Eugene, Oregon).
  • Table 1 shows that DNA was able to effectively adsorb to lot CD056 (Formulation B), with 26.9 ⁇ g out of the initial 50 ⁇ g input being adsorbed to the particles. In contrast, htde DNA was adsorbed to the microparticles utihzed in the experiment described below (Formulation A). Table 1 suggests that a small amount of DNA may have adsorbed to TC339 (4.6 ⁇ g). It is interesting to note that formulations CD056 (B) and TC339 (A) were prepared in identical manners with the sole exception of the polymer used. Hence, the abihty of plasmid DNA to bind is influenced by more subde factors than simply the presence or absence of a cationic component.
  • Adenoviral vectors expressing TB antigens 38-1, DPV, and TbH9 were made by subcloning the reading frame of the antigen into pShutde CMV.
  • the resultant plasmid, pShutde CMV/38-1 was recombined with pAdEasyl in E.r ⁇ /z BJ5183 cells.
  • the recombination event between the two plasmids assembled a chimeric plasmid, pAd38-l containing the complete adenovirus serotype 5 genome with the expression cassette replacing the viral El region.
  • the bacterial portion of pAd38-l was removed by Pad digestion and this DNA was used to transfect human embryonic kidney 293 cells (HEK293) to generate a recombinant adenoviral vector.
  • the virus stock was purified over CsCl gradients and dialyzed against Hepes buffered saline. The biological titer was determined by plating dilutions of the purified virus on HEK293 cells.
  • 1 -IB is a class I restricted (HLA-B44) CD8 + T-cell clone that is specific for the Mycobactenum tuberculosis antigen 38-1 (also known as CFP-10/Mtbl l).
  • 20,000 1-lB T cells were cultured with HLA-matched monocyte-denved dendritic cells (DC, 20,000/well) that had been infected 24 hr before with recombmant adenovirus expressing the 38-1 gene or other control, as described below. Cultures were performed in triplicate in flat-bottomed 96-well microtiter plates.
  • test conditions for treatment of dendritic cells included: (1) 38-1 adenovirus alone (adeno/38-1); (2) adeno/38.1 plus microparticles after 30 minutes incubation at room temperature; (3) adeno/38.1 mixed with pofectamine prior to addition to DC; and (4) peptide 2-11, which is the minimal peptide recognized by these T cells.
  • the DCs were cultured with these stimuli for 2 hr, at which time the supernatant was removed and the cells were washed once with culture medium. The medium was replaced and the cells were cultured overmght in fresh medium containing GM-CSF and IL-4 (20 ng/ml of each). This medium was removed and replaced with medium containing 1-lB T cells to give a final volume of 200 ⁇ l/well. The plates were cultured for an additional 72 hr, when 50 jol of supernatant was removed for determination of IFN- ⁇ levels by ELISA. The plates were then pulsed overnight with t ⁇ tiated thymidine
  • a CD8+ CTL clone termed 1-lB that responds specifically to the Mycobactenum tuberculosis antigen 38-1 was utihzed to compare the presentation of the virally encoded antigen generated by the microparticle-adenovirus formulation to that of adenovirus particles alone.
  • Adenovirus plus Lipofectamine was included as a positive control, as this combination has previously been shown to produce increased levels of infection over virus alone in in vitro systems.
  • Fig. 1 shows the proliferation (thymidine incorporation) responses in this in vitro assay.
  • Figure 1 demonstrates that adenovirus alone was inefficient at stimulating 1-lB cells. The use of Lipofectamine marginally improved the stimulation of 1-lB T cells.
  • microparticle-adenovirus formulation strongly enhanced stimulation of 1-lB T cells such that a multiplicity of infection (MOI) of 1 was more stimulatory than seen with an MOI of 100 with the naked 38-1 adenovirus (Fig. 1).
  • MOI multiplicity of infection
  • IFN- ⁇ production approximately a 300-fold greater MOI was needed to stimulate the 1-lB T cells with naked 38-1 adenovirus compared with adenovirus adsorbed to microspheres (Fig. 2).
  • Example 5 Additional microparticle formulations provide effective vehicles for viral dehvery
  • the TC339 and TC350b give identical results, indicating that the preparation of this microparticle formulation (A) is reproducible. These results also indicate that, while an additional cationic microparticle formulation (CD 165b, Formulation B) is capable of increasing infection and antigen presentation of the DC, it decreases the efficiency of presentation at higher concentrations. There are several possible explanations for this effect, including cell toxicity and steric hindrance of the cellular interaction.
  • the non-cationic microparticles (CD 125, Formulation C) are also capable of enhancing antigen presentation of the Adv-encoded antigen, however, they do so at an efficiency approximately 30-fold lower than TC339 (Formulation A).
  • Example 6 Presentation of virally encoded antigen dehvered as conjugates with microparticles is effective for multiple antigens
  • Example 7 Improved infection of human dendritic cells by adenovirus conjugated to microspheres
  • This example demonstrates dramatic improvement of in vitro infection of human dendritic cells by utilizing adenovirus conjugated to cationic microspheres.
  • Recombinant adenovirus expressing hrGFP (Stratagene) was conjugated to microparticle formulation A (Example 1).
  • Recombinant adenovirus conjugated to microparticles was compared to naked adenovirus and virus pre-incubated with lipofectamine.
  • Virus conjugates were plated with human DC at various MOI for 16 hours at 37°C. Gene expression was monitored by fluorescence microscopy.
  • naked adenovirus does not infect human DC efficiendy at an MOI of 20.
  • Pre-incubation of the virus particles with lipofectamine gready improves infection at this time point ( Figure 7B).
  • conjugation of the adenovirus particles with cationic microspheres shows a dramatic increase in lnfectivity, not only over naked adenovirus, but also over the vurus/hpofectamine combination ( Figure 7C).

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Abstract

La présente invention concerne un vecteur viral conjugué à une microparticule, ledit vecteur viral comprenant un polynucléotide codant un polypeptide hétérologue. Le fait de conjuguer le vecteur viral à la microparticule produit une augmentation spectaculaire de l'efficacité de la réponse immune déclenchée. La présente invention concerne également une méthode d'apport d'un polynucléotide à une cellule qui consiste à mettre en contact la cellule avec un vecteur viral selon l'invention. Dans une forme de réalisation préférée, la cellule est une cellule présentatrice de l'antigène, telle qu'une cellule dendritique. L'invention concerne également un vaccin comprenant un vecteur viral selon l'invention ; une méthode d'apport d'un polynucléotide à un individu, une méthode de stimulation d'une réponse immune chez un individu ; une méthode de traitement du cancer chez un individu ; une méthode d'inhibition de la croissance tumorale chez un individu ; et une méthode de traitement d'une infection chez un individu.
PCT/US2002/000235 2001-01-05 2002-01-07 Microparticules et methodes d'apport de vaccins a l'aide de virus de recombinaison WO2002092132A2 (fr)

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WO2006053871A3 (fr) * 2004-11-16 2006-12-21 Crucell Holland Bv Vaccins multivalents comportant des vecteurs viraux recombinants
EA012037B1 (ru) * 2004-11-16 2009-06-30 Круселл Холланд Б.В. Поливалентные вакцины, содержащие рекомбинантные вирусные векторы
CN101090974B (zh) * 2004-11-16 2011-05-11 克鲁塞尔荷兰公司 包含重组病毒载体的多价疫苗
US8012467B2 (en) 2004-11-16 2011-09-06 Crucell Holland B.V. Multivalent vaccines comprising recombinant viral vectors
JP4838259B2 (ja) * 2004-11-16 2011-12-14 クルセル ホランド ベー ヴェー 組み換えウイルスベクターを含む多価ワクチン
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US8609402B2 (en) 2004-11-16 2013-12-17 Aeras Global Tb Vaccine Foundation Multivalent vaccines comprising recombinant viral vectors
CN102220357B (zh) * 2004-11-16 2013-12-25 克鲁塞尔荷兰公司 包含重组病毒载体的多价疫苗
WO2023008415A1 (fr) 2021-07-27 2023-02-02 ＳＴＡＮＤ Ｔｈｅｒａｐｅｕｔｉｃｓ株式会社 Marqueur peptidique et acide nucléique codant pour celui-ci

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